VACCINE STUDIES: A crucial element in the development of effective prophylactic strategies for AIDS is an experimental animal model in which the course of immunodeficiency virus infection parallels the pathogenesis of the human disease. SIV infection of macaques is a relevant model since it induces an immunodeficiency syndrome in infected macaques that is remarkably similar to human AIDS. Recent studies suggest that SIV-infection is likely to be more representative of human AIDS pathogenesis than SHIV viruses that utilize CXCR4 as their coreceptor and target naive rather than memory CD4+ T cells. During primary viremia in SHIV-infected macaques, massive elimination of CXCR4 naive CD4+ T cells occurred. In contrast, CCR5+ memory CD4+ T cells were selectively depleted in rapidly progressing SIV-infected macaques. Thus SHIV and SIV target different subsets of CD4+ T cells. These differences explain the different pathogenesis of SIV and SHIV. Importantly, in the context of developing an effective vaccine, regimens that suppress SHIV might not protect monkeys against SIV or humans against HIV. The major vaccine effort within my laboratory has been the evaluation of the highly attenuated vaccinia virus Ankara (MVA) strain as a recombinant vector. A cohort of rhesus macaques were immunized with recombinant MVA viruses that expressed either gag-pol alone, env alone, or a combination of gag-pol and env. Macaques expressing the MamuA*01 MHC-Class I allele were used to evaluate cellular immune responses using tetramer assays with the Gag P11C-M epitope. All macaques became infected following intravenous challenge with pathogenic SIVsmE660. However, plasma viremia in each of the groups immunized with MVA-SIV recombinants was significantly reduced as compared to the group vaccinated with nonrecombinant MVA and their survival was prolonged. These data demonstrate that vaccination with MVA-SIV recombinants results in significant protection from high viremia and AIDS. These animals are the focus of follow-up studies to determine the long-term efficacy of such vaccines. All but two vaccinees have progressed to AIDS by 7 years after challenge suggesting that immune escape from such vaccines is a major concern. A new MVA recombinant that expresses the SIVsmE543-3 envelope has been generated for future experiments. This envelope has more neutralization. Therefore, it is a better model for HIV-1 primary isolates. We also evaluated the MVA strategy using a CXCR4-tropic SIV, 89.6P challenge model in rhesus macaques. Four MamuA*01+ macaques immmunized with MVA expressing SIVmac239 gag-pol and MVA expressing HIV/89.6 env were challenged with pathogenic SHIV/89.6P. A significant reduction in set point viremia and partial protection from CD4 lymphocyte depletion was observed. This degree of protection was similar to that observed in macaques immunized with cytokine augmented DNA and DNA prime-MVA boosted macaques. This contrasts with the far less robust protection observed in macaques immunized with a comparable regimen following SIV challenge. Viral escape from cytotoxic T lymphocytes (CTLs) can undermine immune control of human immunodeficiency virus 1. It is therefore important to assess the stability of viral mutations in CTL epitopes after transmission to naive hosts. In collaboration with the Letvin lab, we demonstrated the persistence of mutations in a dominant CTL epitope after transmission of simian immunodeficiency virus variants to major histocompatibility complex-matched rhesus monkeys. Transient reversions to wild-type sequences occurred and elicited CTLs specific for the wild-type epitope, resulting in immunological pressure that rapidly reselected the mutant viruses. These data suggest that mutations in dominant human immunodeficiency virus 1 CTL epitopes may accumulate in human populations with limited major histocompatibility complex heterogeneity by a mechanism involving dynamic CTL control of transiently reverted wild-type virus. A non-integrating mutant, SIVsmD116N clone, was derived from SIVsmE543-3 by introducing an Asp (D) to Asn (N) mutation into the invariant D-116 integrase residue in the catalytic domain. This point mutation completely abolishes viral DNA integration of HIV without affecting other known viral functions such as reverse transcription and nuclear targeting. The SIVsmD116N and wild type SIVsmE543-3 clones were transfected into 293T cells to generate cell free virus and their replication was assessed in CEMx174 cells, macaque PBMC and macaque monocyte-derived macrophages (MDM). While the wild type virus replicated in each of these cell types, no RT activity was observed in cell-free media following infection with the integrase mutant. Alu-PCR confirmed that SIVsmD116N did not integrate into genomic DNA. The SIVsmD116N mutant was able to synthesize viral DNA with almost equal efficiency to wild type virus and viral DNA persisted in macrophages for as long as 30 days. The capacity of a nonintegrating SIV to persistently generate viral products suggests that nonintegrating lentiviral vectors could serve to express protein for vaccine purposes, without the permanency of an integrated retrovirus or disruption of normal cellular genes. PHYLOGENY OF SIV/HIV: The human immunodeficiency viruses, HIV-1 and HIV-2, are members of a large family of primate lentiviruses that have their origins in African primates. Each of the human viruses arose following cross-species transmission from a naturally-infected primate to humans SIVsm from sooty mangabey monkeys for HIV-2 and SIVcpz from chimpanzees for HIV-1. The goal of this project is to molecularly characterize novel SIV isolates from wild-caught African monkeys. We initiated these studies by characterizing SIV from a sooty mangabey (SIVsm), SIV from three of the species of African green monkeys (vervets, grivets and tantalus), SIV from Sykes monkeys and SIV from L'Hoest monkeys (C. lhoesti). We cloned and sequenced the entire genome of SIVrcm from a wild caught redcapped mangabey from Nigeria and demonstrated that this virus is a complex recombinant. SIVmnd-2 was a mosaic genome, highly related to SIVrcm in the 5' portion of the genome and to SIVmnd-1 in the 3' portion of the genome. Thus it appears that SIVrcm, while in itself a recombinant, is also the source of a portion of the SIVmnd-2 genome. These data are suggestive of cross-species transmission and recombination in the evolution of these viruses. We also characterized a SIVdrl and novel SIVmnd-type 2 isolate. These studies revealed that both of these viruses are recombinants between a SIVrcm-like virus and a SIVlhoest like virus and share a common recombination point. These data are consistent with a complex history of recombination and cross-species transmission involving SIV in drills, mandrills, redcapped mangabeys and L'hoest monkeys. As an adjunct to a pathogenesis study, we characterized the envelope genes of SIVagm isolated from PBMC of a cohort of African green monkeys (vervet) imported from Tanzania. This analysis revealed that these viruses clustered with other known SIVagmVer sequences. The evolution of SIV in the cerebral spinal fluid (CSF) was compared with the virus that evolved in the plasma of two rhesus macaques which developed SIV encephalitis. While the virus in the CSF and plasma were similar during primary infection, distinct substitutions were observed sequentially in the two compartments. These findings are consistent with compartmentalization between the brain and blood during development of neuro-AIDS and the evolution of viruses with distinct genotypes and potentially distinct biological phenotypes in the brain.